Restriction Enzymes 187
bond, polarization of the P-O bond by Mg 2÷ and protonation of the leaving
group by a Mg 2÷ bound water molecule. Site-directed-mutagenesis
experiments have made it possible to define in other restriction
enzymes (e.g., BamHI [213]) amino acid residues involved in catalysis.
The observation that many restriction enzymes, depending on
sequence context and reaction conditions, cleave the two strands of the
DNA in separate binding events has given rise to the assumption that
the accuracy of restriction enzymes can be increased by proofreading
using DNA ligase. This was shown to be the case in vitro and in vivo
(214). The requirement for proofreading could explain why all restric-
tion enzymes produce 5' phosphorylated ends and free 3'-ends, because
these are the substrates for DNA-ligase.
The purification of restriction enzymes is comparatively easy when
their genes are cloned in expression vectors. A further simplification
can be achieved when their genes are fused with DNA sequences
coding for affinity "tags." For example, C-terminal His 6 tags allow
one-step-purification of the EcoRI:His 6 fusion protein. The resulting
preparation is homogeneous, fully active, and devoid of contaminat-
ing nonspecific nuclease activities (215).
Assaying the enzymatic activity of restriction enzymes has been
simplified considerably by a recently developed continuous spectro-
photometric assay, which takes advantage of the hyperchromic effect
produced by dissociation of the product double strands after cleavage
of short oligodeoxynucleotides (216).
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viruses. J. Bacteriol. 65, 113-121. - Arber, W. and Dussoix, D. (1962) Host specificity of DNA produced by
Escherichia coli. I: Host controlled modification of bacteriophage ~,. J. Mol.
Biol. 5, 18-29. - Meselson, M. and Yuan, R. (1968) DNA restriction enzyme from Escherichia
coli. Nature 217, 1110-1114. - Linn, S. and Arber, W. (1968) In vitro restriction of phage fd replicative
form. Proc. Natl. Acad. Sci. USA 59, 1300-1305. - Smith, H. O. and Wilcox, K. W. (1970) A restriction enzyme from
Haemophilus influenzae. I: Purification and general properties. J. Mol. Biol.
51, 379-391. - Roulland-Dussoix, D. and Boyer, H. W. (1969) The Escherichia coli B restric-
tion endonuclease. Biochim. Biophys. 195, 219-232.